CN101007848A - Long chain human insulin-like growth factor(LR3IGF-1) and its preparing process and application method - Google Patents
Long chain human insulin-like growth factor(LR3IGF-1) and its preparing process and application method Download PDFInfo
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Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
本发明涉及一种新的长链人胰岛素样生长因子，其具有促进细胞和/或组织生长、增殖和/或再生的功能。另外，本发明还涉及制备该长链人胰岛素样生长因子的方法，以及该生长因子在药品、食品、保健品和化妆品等方面的应用。The present invention relates to a novel long-chain human insulin-like growth factor, which has the function of promoting cell and/or tissue growth, proliferation and/or regeneration. In addition, the present invention also relates to a method for preparing the long-chain human insulin-like growth factor, and the application of the growth factor in medicine, food, health products, cosmetics and the like.
Description
Technical field
The invention belongs to biological recombinant technology field.More specifically, (Long R3 Insulin-like growth factor-1 abbreviates LR herein as to the present invention relates to a kind of new Long IGF-1 R3
3IGF-1).In addition, the invention still further relates to the method for this Long IGF-1 R3 of preparation, and this somatomedin is in the application of aspects such as medicine, food, healthcare products and makeup.
Background technology
Human insulin-like growth factor is the factor of regulating normal human cell's growth, has inducing or improves tissue growth, propagation and/or regenerated effect.Human insulin-like growth factor promotes the growth of human body by the mediating growth hormone, therefore as cellular replication, bone growth and the other process relevant with growth all be subjected to the human insulin-like growth factor level influence (referring to J.Clin.Endocrinol.Metab, 192 (75): 183-188).Human insulin-like growth factor can promote the growth of mammalian cell in serum free medium, for example, it can stimulate the propagation (J.Biol.Chem. of thyroid cell, 264:18485-18488), can also act synergistically with some other somatomedin, as promote the damage of soft tissue and mesenchymal tissue reparation (Endocrinology, 131:2399-2403).
Therefore, rhIGF-1 has been widely used in preparing the aspects such as medicine, food, healthcare products and makeup that promote the cell growth at present.Chinese patent application CN1251531A discloses and can utilize Induced by IGF or improve tissue growth, propagation or regenerated method, especially utilizes rhIGF-1 to promote the growth of inner ear tissue; Chinese patent application CN1204263A has disclosed and can utilize rhIGF-1 treatment brain or spinoneural degenerative disease, as alzheimer's disease (Alzheimer ' s Disease), Parkinson's disease (Parkinson ' s Disease) etc.Aspect the treatment diabetes, also there is lot of documents to disclose and utilizes rhIGF-1 to treat put into practice (referring to Chinese patent application CN1387440A, the CN1384201A etc.) of aspects such as I type, II type and development of insulin resistance diabetes.Can extracting from cell of human insulin-like growth factor also can prepare by genetically engineered, and commercial human insulin-like growth factor, the genetically engineered recombinant human insulin-like growth factor of Cephalon company have been arranged at present.
In addition, people are also at the derivative and the analogue of active development human insulin-like growth factor.The U.S. Pat 5679771A of Gropep company discloses a series of specific long-chain rhIGF-1s, wherein Long IGF-1 R3 (LR
3IGF-1) the human insulin-like growth factor derivative of being made up of 83 amino-acid residues, molecular weight is about 9000Da.This LR
3IGF-1 is by on the human insulin-like growth factor sequence amino-acid residue Glu being substituted by Arg and having added 13 amino acid and the polypeptide that forms at the N-terminal of IGF-1.As described in the patent of Gropep company, this LR
3IGF-1 can keep the biologic activity of human insulin-like growth factor, promotes cell, tissue growth, propagation or regeneration, thereby can substitute human insulin-like growth factor and use at aspects such as medicine, food, healthcare products and makeup.The inventor has obtained a kind of new Long IGF-1 R3 through long-term and arduous research.Although LR of the present invention
3IGF-1 also is made up of 83 amino-acid residues, but its sequential structure can't from open source literature, derive, with the LR of Gropep company
3IGF-1 compares only has 73% identity, has complete independent intellectual property right; And more beat all be that it also has the LR than Gropep company
3The biologic activity that IGF-1 is higher.
Summary of the invention
The object of the present invention is to provide a kind of new Long IGF-1 R3, it can promote cell, tissue growth, propagation or regeneration, and the Long IGF-1 R3 compared to prior art even have higher biologic activity.The present invention also aims to provides nucleic acid molecule and carrier, the host cell of the described Long IGF-1 R3 of coding and utilizes genetic engineering technique to prepare the method for described Long IGF-1 R3.In addition, the application that the present invention also aims to provide the pharmaceutical composition of this Long IGF-1 R3 and be used for promoting medicine, protective foods and/or the makeup of cell proliferation in preparation.
Particularly, aspect first, the invention provides a kind of Long IGF-1 R3, its aminoacid sequence
A) shown in the sequence in the sequence table 1; Or
B) be to the sequence shown in the sequence in the sequence table 1 add, lack and/or replace one or several amino-acid residue and aminoacid sequence, and described Long IGF-1 R3 can promote cell proliferation.
The aminoacid sequence of the Long IGF-1 R3 of preferred first aspect of the present invention is shown in the sequence in the sequence table 1.The inventor has found the Long IGF-1 R3 of first aspect of the present invention through arduous research, and surprisingly, the preferred Long IGF-1 R3 of the present invention is than the disclosed LR of prior art
3IGF-1 has higher biologic activity.
The Long IGF-1 R3 of first aspect of the present invention also can be the varient of the Long IGF-1 R3 of aminoacid sequence shown in the sequence in the sequence table 1, the aminoacid sequence of described varient is to add on the basis of the sequence shown in the sequence 1 in sequence table, the disappearance and/or replace one or several amino-acid residue and aminoacid sequence, preferably add, the disappearance and/or replace one to five and, be more preferably interpolation, disappearance and/or replace one to three and, and described Long IGF-1 R3 still can promote cell proliferation.Those skilled in the art know, import expression vector by the coding gene sequence of change known peptide and with it, can prepare replacement, added or lack the polypeptide of amino-acid residue, these methods extensively are recorded in " molecular cloning experiment guide " documents well known in the art such as (Beijing: Science Press, 2002).In the amino-acid residue that replaces, preferably be substituted by other amino acid with original acid residue side chain similar performance, thus the easier original function activity that kept.The amino acid of side chain similar performance has hydrophobic amino acid (A respectively, I, L, M, F, P, W, Y, V), hydrophilic amino acid (R, D, N, C, E, Q, G, H, K, S, T), amino acid (the G of aliphatic lateral chain, A, V, L, I, P), amino acid (the S of hydroxyl side chain, T, Y), amino acid (the C of sulfur atom-containing side chain, M), amino acid (the D that contains carboxylic acid and amide side chains, N, E, Q), amino acid (the R that contains the basic group side chain, K, H), amino acid (the H that contains the aromatic series side chain, F, Y, W).Existed at present a large amount of known checks to promote the experimental technique of ability of cell proliferation, those skilled in the art can preferably select the Long IGF-1 R3 with above-mentioned functions by the described concrete experimental technique of the embodiment of the invention according to definite biological function and/or the activity that replaces, adds or lack the aminoacid sequence that gets of these methods from the aminoacid sequence through adding, lack or replacing.The biologic activity of the preferred varient of the present invention is not less than 50% of the Long IGF-1 R3 biologic activity of aminoacid sequence shown in the sequence in the sequence table 1, more preferably is not less than 80%, more preferably is not less than 90%, most preferably is identical.
Aspect second, the invention provides a kind of nucleic acid molecule, its code book is invented the described Long IGF-1 R3 in first aspect.Nucleic acid molecule of the present invention can be a dna form, also can be rna form, preferably dna form.Dna form comprises the cDNA of natural cDNA and synthetic, and DNA can be coding strand or template strand.By routine techniques, as the method for PCR method, recombination method or synthetic, those skilled in the art can be easy to obtain nucleic acid molecule or its fragment of Long IGF-1 R3 of the present invention.These sequences transform or are transfected into corresponding cell again in case acquisition just can be cloned into carrier with it, breed by the host cell of routine then, therefrom separate obtaining a large amount of nucleic acid molecule.The nucleotide sequence of preferred nucleic acid molecule of the present invention is shown in the sequence in the sequence table 2.This preferred sequence can be expressed in yeast.
Aspect the 3rd, the invention provides a kind of carrier, it contains second described nucleic acid molecule in aspect of the present invention.Term herein " expression vector ", " carrier " can be replaced use alternately at this, are meant bacterial plasmid, clay, phagemid, yeast plasmid, vegetable cell virus, animal virus and other various virus vector commonly used in this area.The carrier that is suitable among the present invention includes but not limited to: the carrier (prokaryotic expression carrier) of expressing usefulness in bacterium, in yeast, express the carrier of usefulness (as pichia vector, debaryomyces hansenii carrier etc.), baculovirus vector in expressed in insect cells, in mammalian cell, express the carrier (vaccinia virus vector of usefulness, retroviral vector, adenovirus carrier, adeno-associated virus carrier etc.), the various carriers of in plant, expressing the plant viral vector of usefulness and in mammal galactophore, expressing usefulness.In a word, as long as can make second described nucleic acid molecule in aspect of the present invention stable duplicating in host cell, any plasmid and carrier all can use.The preferred expression carrier comprises selectable marker gene, as ampicillin resistance gene, tetracycline resistance gene, kalamycin resistance gene, streptomycin resistance gene, the chloramphenicol resistance gene of bacterium; Saccharomycetic neomycin resistance gene, Zeocin resistant gene, saccharomycetic defective selection marker, as His, Leu, auxotrophs such as Trp; Eukaryotic neomycin resistance gene, Zeocin resistant gene, dihydrofolate reductase gene and fluorescent protein labeling gene etc.Carrier of the present invention is preferably eukaryotic vector, and in the specific embodiment of the present invention, it is pPICZ-LR
3IGF-1, the i.e. expression plasmid of nucleotide sequence shown in the sequence 2 in clone's ordered list on pPICZ α A plasmid.Those skilled in the art can utilize a series of technology such as DNA recombinant technology, make up the dna sequence dna contain proteins encoded of the present invention, suitable expression vector of transcribing with particular element such as translational control sequence, promotor and selected markers.Above-mentioned carrier can be used to transform, the transfection proper host cell, so that obtain needed Long IGF-1 R3.
Aspect the 4th, the invention provides a kind of host cell, it is characterized in that described cell contains the described carrier of third aspect of the present invention, perhaps described cell gets with the described nucleic acid molecule conversion in second aspect of the present invention or transfection.Host cell can be a prokaryotic cell prokaryocyte, also can be eukaryotic cell, as, bacterial cell, yeast cell, vegetable cell, insect cell, mammalian cell etc.Host cell promptly constitutes through engineering approaches cell or cell strain after transforming or transfection contains the gene order of proteins encoded of the present invention, can be used for producing desirable proteins.Those skilled in the art can select appropriate carriers, host cell rightly, and how know carrier high-efficiency ground is transformed or is transfected in the host cell, method therefor includes but not limited to: Calcium Chloride Method, electroporation are used for bacterial cell, electroporation and protoplastis fusion method are used for yeast cell, and liposome, coprecipitation of calcium phosphate, electro fusion method and microinjection are used for eukaryotic cells such as mammalian cell.Preferred host cell of the present invention is Pichia pastoris yeast expression bacterial strain GS115.This preferred host cell energy efficient secretory expression Long IGF-1 R3, thus the work of downstream separation purifying simplified greatly.
Aspect the 5th, the invention provides the composition that is used to promote cell proliferation, it comprises described Long IGF-1 R3 in first aspect of the present invention and pharmaceutically acceptable carrier.Described pharmaceutical composition can promote cell proliferation.To those skilled in the art, there have been a lot of known method protein or polypeptide active composition can be become pharmaceutical composition and makeup with pharmaceutically acceptable preparing carriers.Pharmaceutically acceptable carrier used herein refers to nontoxic weighting agent, vehicle, thinner or other pharmaceutical adjuncts, and they are compatible with activeconstituents.Using pharmaceutically acceptable preparing carriers pharmaceutical composition and makeup is known for those of ordinary skills.Composition of the present invention comprises the described Long IGF-1 R3 in first aspect of the present invention as activeconstituents, this Long IGF-1 R3 and pharmaceutically acceptable carrier (as vehicle well known to those of ordinary skill in the art, thinner etc.) are combined, be mixed with various preparations, be preferably solid preparation and liquid preparation.Preferred preparation of the present invention is a unit dosage form, as formulations such as tablet, ointment, pill, capsule (comprise and continue to discharge or postpone releasing pattern), pulvis, suspensoid, granule, tincture, syrup, emulsion agent, suspension, injections, thereby be fit to various form of medication, the form of medication of for example oral, transdermal, non-enteron aisle injection, mucous membrane, muscle, intravenously, subcutaneous, intraocular, intracutaneous etc.Composition of the present invention is especially preferably made spray, ointment or injection.Preferred composition of the present invention comprises damping fluid (as phosphate buffered saline buffer, Tris-HCl damping fluid) or physiological saline.For example, for the epidermis of rat damage, the physiological saline that contains Long IGF-1 R3 of the present invention can promote wound healing.
Aspect the 6th, the invention provides the described Long IGF-1 R3 in first aspect of the present invention is used for promoting cell and/or tissue growth, propagation and/or regenerated medicine, protective foods and/or makeup in preparation application.Long IGF-1 R3 of the present invention has the effect that promotes cell fission, propagation, can be used for inducing or improving patient's tissue growth, propagation or regenerated level, treat corresponding disease, for example brain or spinoneural degenerative disease, as alzheimer's disease, Parkinson's disease etc., can be used in and treat and/or prevent the disease that aspects such as disorder are regulated in the metabolism of carbohydrate equal energy source, disease as aspects such as treatment and prevention carbohydrate metabolism or metabolism of fat, particularly diabetes comprise I type, II type and development of insulin resistance diabetes etc.In addition, healthy people takes a certain amount of Long IGF-1 R3 of the present invention and also helps to keep its normal cell fission, proliferation function, helps to improve sub-health state, keeps its abundant vigor.A certain amount of Long IGF-1 R3 of the present invention of healthy people's external application helps to keep division, the propagation of its skin cells, improves outside image, has the effect of beauty treatment.
For medicine, the dosage of administration and form are generally determined according to patient's particular case (as age, body weight, sex, the state of an illness, sick time, physical appearance etc.) by the doctor.Generally speaking, the dosage of administration is 0.001～100mg/kg weight in patients, is preferably 0.01～10mg/kg, more preferably 0.1～1mg/kg.Form of medication can determine according to the formulation of various pharmaceutical preparations, and form of medication such as that the form of medication that is fit to has is oral, transdermal, non-enteron aisle injection, mucous membrane, muscle, intravenously, subcutaneous, intraocular, intracutaneous preferably use spray or ointment.The described Long IGF-1 R3 in first aspect of the present invention can use as medicine, protective foods and/or makeup separately, also can add in other medicines, protective foods and/or the makeup and use.For example, nanometer goods of the present invention can add fit applications in conventional beverage, food or the makeup to.
Aspect the 7th, the invention provides the method for preparing first described Long IGF-1 R3 in aspect of the present invention, it comprises, with the described Long IGF-1 R3 in the present invention first aspect of described host cell expression the present invention, the 4th aspect, the described Long IGF-1 R3 of separation and purification then.The engineering cell that obtains can be cultivated by ordinary method, be induced and express needed albumen, comprises fermenting process and purifying process.Above-mentioned expressed proteins can be in cell, on the cytolemma or be secreted into cell pericentral siphon, extracellular.As required, can utilize physics, chemistry and other biological characteristics of fusion rotein, carry out separation and purification.Method includes but not limited to: split bacterium (ultrasonic wave is split bacterium, infiltration pressure break bacterium), centrifugal, saltout molecular sieve chromatography, ion-exchange chromatography, adsorption chromatography (affinity chromatography, metal chelate affinity chromatography), reverse chromatograms, high performance liquid chromatography, capillary electrophoresis, the sex change of isoelectrofocusing of preparation property and routine, renaturation processing etc., these methods all are well-known to those skilled in the art.In order to obtain Long IGF-1 R3 efficiently,, preferably in the method aspect the 7th of the present invention, use carrier pPICZ-LR especially for making things convenient for the downstream separation purification process
3IGF-1 transfection Pichia pastoris yeast expression bacterial strain GS115, wherein carrier pPICZ-LR
3IGF-1 contains second described nucleic acid molecule in aspect of the present invention.In addition, because Long IGF-1 R3 of the present invention can be secreted in the substratum of fermented yeast cell, preferably in the method aspect the 7th of the present invention, the step of the described Long IGF-1 R3 of separation and purification comprises ultrafiltration.Wherein, those skilled in the art can select for use suitable ultra-filtration membrane to filter and/or hold back the Long IGF-1 R3 of corresponding molecular weight.
The present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is the same excessively in this article for their full text.
For the ease of understanding, below will describe in detail the present invention by specific embodiment.It needs to be noted that these descriptions only are exemplary descriptions, do not constitute limitation of the scope of the invention.According to the argumentation of this specification sheets, many variations of the present invention, change have been obviously all concerning one of ordinary skill in the art.
Embodiment
The following stated experimental technique does not specify, all according to " molecular cloning experiment guide ", the third edition,, Science Press (Beijing) in 2002) described method carries out.
Embodiment 1
LR of the present invention
3The clone of IGF-1 gene
Entrust the synthetic LR of the present invention of pool, Shanghai weighing apparatus Bioisystech Co., Ltd by commercial sources
3The IGF-1 gene also is built into the expression plasmid carrier.Clone's process is carried out with reference to the appended operational guidance of T4 polynucleotide kinase, T4 dna ligase, restriction enzyme of " molecular cloning experiment guide " and used TaKaRa company (Japan), and concise and to the point process is as follows:
Pass through automatic dna synthesizer, 5 nucleic acid fragments (sequence is respectively shown in the sequence 3,4,5,6,7 of sequence table) of synthetic IGF-1 gene, with T4 polynucleotide kinase (available from TaKaRa company) 5 ' end of these 5 nucleic acid fragments is carried out phosphorylation, wait then behind these 5 nucleic acid fragments of mixed in molar ratio in 65 ℃ of sex change 5 minutes, annealing is cooled to 16 ℃, adds T4 dna ligase (available from TaKaRa company) and connects 12 hours.Then, get the above-mentioned connection product of 1 μ L and in 50 μ L reaction volumes, carry out PCR, wherein forward primer (has been introduced EcoR I restriction enzyme site) shown in the sequence 8 of sequence table, reverse primer (introduced Xba I restriction enzyme site) shown in the sequence 9 of sequence table, reaction conditions was: with 94 ℃ of sex change 4 minutes, extend with 94 ℃ of sex change 30 seconds, 63 ℃ of annealing 30 seconds and 72 ℃ then and carried out 35 circulations in 30 seconds, extended 4 minutes with 72 ℃ at last and be cooled to 4 ℃.
The above-mentioned PCR product of agarose gel electrophoresis, reclaim the fragment of about 260bp size, with EcoR I and this fragment of Xba I double digestion, and be connected with the T4 dna ligase through the pPICZ of these two endonuclease digestions α A plasmid (available from Invitrogen company), be transformed among the intestinal bacteria Top10F '.Choose positive colony, extracting goes out plasmid wherein, weighs Bioisystech Co., Ltd with plasmid (the called after pPICZ-LR that builds by the pool, Shanghai after sequence verification is errorless
3IGF-1) return.
Embodiment 2
LR
3The structure of IGF-1 yeast secreted expression bacterial strain
According to the yeast conversion operational guidance of " molecular cloning experiment guide " and Invitrogen company, with pPICZ-LR
3IGF-1 imports Pichia pastoris yeast expression bacterial strain GS115 (available from Invitrogen company).Process is as follows:
(1) linearizing pPICZ-LR
3IGF-1 plasmid DNA: in 100 μ l reaction systems, add 20 μ gpPICZ-LR
3IGF-1 plasmid DNA and 20 PmeI of unit enzymes (available from New England Biolabs) were cut 3 hours in 37 ℃ of enzymes, added 2 μ l 200mM EDTA, termination reaction.Then, add 100 μ l deionized waters, add equal-volume phenol/chloroform, the thermal agitation mixing, 13000rpm high speed centrifugation 10 minutes changes supernatant liquor over to new pipe, add 1/10 volume 3M sodium-acetate, add 2.5 times of volume dehydrated alcohols again, placed 1 hour in-70 ℃ behind the mixing, again 4 ℃ with 13000rpm centrifugal 15 minutes, discard liquid, once with 80% washing with alcohol, dry in the air, to precipitate and be dissolved in again in the 10 μ l deionized waters, place 0 ℃, stand-by.
(2) preparation yeast competent cell: in 5ml YPD substratum (YPD culture medium preparation: dissolving 10g yeast extract and 20g peptone in the 900ml water, behind the autoclaving, to be cooled during to 55～60 ℃, add 100ml 20% glucose solution) middle inoculation Pichia pastoris yeast expression bacterial strain GS115,30 ℃ of shaking culture are spent the night, in the new YPD substratum of the 500ml that transfers next day, 30 ℃ are continued to cultivate, until OD
600Reach till 1.3～1.5.4 ℃ with 1500rpm centrifugal 8 minutes, abandoning supernatant, the thalline of collecting precipitation, the 500ml aseptic deionized water that the adds precooling thalline that suspends again.Then, recentrifuge, 4 ℃ with 1500rpm centrifugal 8 minutes, abandoning supernatant, the thalline of collecting precipitation is with the aseptic deionized water of the 250ml precooling thalline that suspends again.Then, at 4 ℃ of thalline, again with the aseptic deionized water of the 20ml precooling thalline that suspends again with centrifugal 8 minutes collecting precipitations of 1500rpm.At last, at 4 ℃ of thalline, use the 1M sorbyl alcohol suspension thalline of 1ml precooling again with centrifugal 8 minutes collecting precipitations of 1500rpm.This competent cell for preparing is placed 0 ℃, stand-by.
(3) saccharomycetic electricity merges: get above-mentioned competent cell of 80 μ l and the above-mentioned linearizing pPICZ-LR of 5 μ l
3The IGF-1 plasmid DNA mixes, and the 0.2cm electricity that changes 0 ℃ of precooling over to merges in the cup, places 5 minutes in 0 ℃.Shock by electricity then, wherein Dian Ji condition is as follows: 1500V, 25 μ F, 20 Ω.The 1M sorbyl alcohol that adds the 1ml precooling then immediately changes over to after mixing in the 15ml test tube, hatches 2 hours in 30 ℃.Get 200 μ l and be laid on the YPDS flat board (preparation of YPDS flat board: dissolving 10g yeast extract, 20g albumen, 182.2g sorbyl alcohol and 20g agar in the 900ml water that contains different concns Zeocin respectively, behind the autoclaving, to be cooled during to 55～60 ℃, the Zeocin that adds 100ml 20% glucose solution and 1ml 250mg/ml, pave plate) on, be inverted, cultivated 2 days for 30 ℃, the picking positive colony, after fermentation and SDS-PAGE electrophoresis checking (process is substantially with embodiment 3) were errorless, this clone was expression LR of the present invention
3The recombinant secretor expression strain of IGF-1.
Embodiment 3
LR
3The fermentation of IGF-1 and purifying
With the LR that obtains among the embodiment 2
3The recombinant secretor expression strain of IGF-1 is inoculated in the 100ml BMGY substratum, and 30 ℃ of shaking culture 16～18 hours are worked as OD
600In the time of between reaching 2～6, with 3000g centrifugal 8 minutes, abandon supernatant, with suspend again sedimentary thalline and add 100% methyl alcohol 0.1ml of 20ml BMGY substratum to final concentration 0.5%, continued shaking culture 72 hours in 30 ℃, during added 100% methyl alcohol 0.1ml every 24 hours.
Then, with 3000g centrifugal 8 minutes, keep fermented supernatant fluid, discard precipitation.Slowly add the ammonium sulfate powder in above-mentioned fermented supernatant fluid, make the ammonium sulfate saturation ratio reach 80%, salt precipitation goes out wherein polypeptide and protein thus.Then, with the centrifugal liquid 15 minutes of saltouing of 7000g, abandon supernatant, precipitation is with the Tris-HCl dissolving of 10mL 20mM pH8.0.Then carry out 2 ultrafiltration: the ultrafiltration molecular weight cut-off is that the ultra-filtration membrane of 100000Da (available from Millopore, Ultrapore), keeps filtrate first; With the filtrate molecular weight cut-off be then 1000Da (available from Millopore, ultra-filtration membrane Ultrapore) carries out ultrafiltration, discards filtrate, washs this ultra-filtration membrane with the Tris-HCl of 10mL 20mM pH8.0, thus the polypeptide that the stripping ultra-filtration membrane is held back.Dissolution fluid is carried out the SDS-PAGE electrophoresis and measures the absorbancy at 280nm place.The result shows, compare with the refined solution behind the blank strain fermentation that obtains after the pPICZ α A transfection, the dissolution fluid of hIGF-1 of the present invention has tangible band on the position about the 9kDa on the electrophorogram, its amount accounts for more than 95% of total protein, i.e. the LR of the present invention of purifying acquisition
3The purity of IGF-1 is passed through OD greater than 95%
280Value obtains LR of the present invention
3The whole productive rate of IGF-1 is 0.2～0.3 grams per liter fermented liquid.
Embodiment 4
LR of the present invention
3The mensuration of the short cell-proliferation activity of IGF-1
Adopt conventional mtt assay to measure LR of the present invention
3The LR of IGF-1, Gropep company
3The short proliferation function of IGF-1 and blank pair cell.Detailed process is as follows: with the DMEM nutrient solution that contains 10% foetal calf serum Balb-3T3 cell (can be numbered ATCC20864 available from U.S. ATCC) is diluted to 1.5*10
4Individual cell/mL.The above-mentioned enchylema of 200 μ L is inoculated in every hole respectively on 96 well culture plates, in 37 ℃, 5%CO
2Cultivated 24 hours.Then, inhale the nutrient solution that goes in every hole, add 200 μ L are diluted to different concns with the DMEM nutrient solution that contains 0.4% foetal calf serum LR of the present invention respectively
3The LR of IGF-1, Gropep company
3HIGF-1 (available from Gropep company) and blank (blank is the DMEM nutrient solution that contains 0.4% foetal calf serum) liquid is in 37 ℃, 5%CO
2Cultivated 24 hours.The MTT liquid that then adds 20 μ L5mg/mL to every hole is in 37 ℃, 5%CO
2Continue to cultivate 4 hours.Inhale the nutrient solution that goes in every hole then, add 150 μ LDMSO (available from Sigma company), placed 1 hour for 37 ℃.Because viable cell can change into tetrazolium salts can be by the coloured product of DMSO dissolved, its output is directly proportional with viable cell quantity, therefore detects the absorbancy at 490nm place with enzyme-linked immunosorbent assay instrument, can estimate viable count.The result is as shown in table 1, with respect to blank, and LR of the present invention
3The LR of IGF-1 and Gropep company
3IGF-1 can both significantly promote cell proliferation, and LR of the present invention
3The activity of IGF-1 more is better than the LR of Gropep company
3IGF-1.
Table 1 LR of the present invention
3The LR of IGF-1 and Gropep company
3The short proliferation function of IGF-1 pair cell
| Concentration | OD 490 | |
| Blank | / | 0.172±0.011 |
| LR of the present invention 3IGF-1 | 10ng/mL | 0.218±0.017 |
| 50ng/mL | 0.240±0.015 | |
| The LR of Gropep company 3IGF-1 | 10ng/mL | 0.200±0.011 |
| 50ng/mL | 0.233±0.007 |
Embodiment 5
LR of the present invention
3The mensuration of the short skin wound healing effect of IGF-1
Prepare the burned rats model according to ordinary method, measure LR of the present invention
3IGF-1 is to the promotion healing effect of rat skin wound.Make Wistar rat (available from Shanghai The 2nd Army Medical College animal center) suck etherization, shave clean rat back, and slough hair with depilatory cream.After 24 hours (promptly at the 1st day), be that the skin biopsy of 5mm is made full thickness (being that epidermis adds dermis thickness) wound with trepan with diameter on the skin of back of every rat, and measure wound area.Fixedly rat will contain 1 μ g/mL LR of the present invention respectively
3The LR of IGF-1, Gropep company
3The physiological saline 50 μ L of IGF-1 or blank (blank is a physiological saline) drip on the rat wound, treat to unclamp rat after the seasoning, so carry out twice and measured wound area in one day.The result is as shown in table 2, with respect to blank, and LR of the present invention
3The LR of IGF-1 and Gropep company
3IGF-1 can both promote skin wound healing, reduces wound area, and LR of the present invention
3The activity of IGF-1 more is better than the LR of Gropep company
3IGF-1.
Table 2 LR of the present invention
3The LR of IGF-1 and Gropep company
3IGF-1 is to the promoter action of skin wound healing
| Skin wound area (%) | |||
| The 1st day | The 3rd day | The 6th day | |
| Blank | 100 | 91 | 72 |
| LR of the present invention 3IGF-1 | 100 | 80 | 52 |
| The LR of Gropep company 3IGF-1 | 100 | 86 | 57 |
Sequence table
＜110〉Zhu Chenggang
＜120〉Long IGF-1 R3 (LR3 IGF-1) and methods for making and using same thereof
<160>9
<210>1
<211>83
<212>PRT
<213>Homo sapiens
<400>1
Met Phe Pro Ala Met Pro Leu Ser Ser Leu Phe Val Asn Gly Pro Arg
1 5 10 15
Thr Leu Cys Ala Val Asp Leu Val Glu Ile Val Gln Phe Val Cys Gly
20 25 30
Asp Lys Gly Phe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser Ser
35 40 45
Arg Arg Ala Pro Gln Thr Gly Leu Ala Glu Asp Cys Cys Phe Lys Thr
50 55 60
Cys Asp Leu Lys Lys Ile Asp Met Tyr Cys Val Pro Leu Arg Pro Val
65 70 75 80
Arg Thr Ala
<210>2
<211>252
<212>DNA
<213>Homo sapiens
<400>2
atgttccccg ccatgcccct gagcagcctg ttcgtgaacg gcccccgcac cctgtgcgcc 60
gtggacctgg tggagatcgt gcagttcgtg tgcggcgaca agggcttcta cttcaacaag 120
cccaccggct acggcagcag cagccgccgc gccccccaga ccggcctggc cgaggactgc 180
tgcttcaaga cctgcgacct gaagaagatc gacatgtact gcgtgcccct gcgccccgtg 240
cgcaccgcct ga 252
<210>3
<211>85
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic
<400>3
atgttccccg ccatgcccct gagcagcctg ttcgtgaacg gcccccgcac cctgtgcgcc 60
gtggacctgg tggagatcgt gcagt 85
<210>4
<211>14
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic
<400>4
cacacgaact gcac 14
<210>5
<211>79
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic
<400>5
tcgtgtgcgg cgacaagggc ttctacttca acaagcccac cggctacggc agcagcagcc 60
gccgcgcccc ccagaccgg 79
<210>6
<211>14
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic
<400>6
ggccaggccg gtct 14
<210>7
<211>88
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic
<400>7
cctggccgag gactgctgct tcaagacctg cgacctgaag aagatcgaca tgtactgcgt 60
gcccctgcgc cccgtgcgca ccgcctga 88
<210>
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic
<400>8
ggaattcatg ttccccgcca tgcccct 27
<210>9
<211>27
<212>DNA
＜213〉artificial sequence
<220>
＜223〉synthetic
<400>9
ctctagatca ggcggtgcgc acggggc 27
Claims (12)
1, a kind of Long IGF-1 R3, its aminoacid sequence
A) shown in the sequence in the sequence table 1; Or
B) be to the sequence shown in the sequence in the sequence table 1 add, lack and/or replace one or several amino-acid residue and aminoacid sequence, and described Long IGF-1 R3 can promote cell proliferation.
2, the described Long IGF-1 R3 of claim 1, its aminoacid sequence is shown in the sequence in the sequence table 1.
3, a kind of nucleic acid molecule, its coding claim 1 or 2 described Long IGF-1 R3s.
4, the described nucleic acid molecule of claim 3, its nucleotide sequence is shown in the sequence in the sequence table 2.
5, a kind of carrier, it contains claim 3 or 4 described nucleic acid molecule.
6, a kind of host cell is characterized in that, described cell contains the described carrier of claim 5, and perhaps described cell is used claim 3 or 4 described nucleic acid molecule conversions or transfection and got.
7, the described host cell of claim 6, it is a yeast.
8, a kind of composition that is used to promote cell proliferation, it comprises claim 1 or 2 described Long IGF-1 R3s and pharmaceutically acceptable carrier.
9, claim 1 or 2 described Long IGF-1 R3s are used for promoting the application of cell and/or tissue growth, propagation and/or regenerated medicine, protective foods and/or makeup in preparation.
10, the described application of claim 9, it is applied as the application that is used for promoting medicine, protective foods and/or the makeup of cell proliferation in preparation, is preferably the application that is used for promoting the makeup of cell proliferation in preparation.
11, the method for preparing claim 1 or 2 described Long IGF-1 R3s, it comprises, with claim 6 or 7 described host cell expression claims 1 or 2 described Long IGF-1 R3s, the described Long IGF-1 R3 of separation and purification then.
12, the described method of claim 12, wherein purification procedures comprises ultrafiltration.
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| CNB2007100034188A CN100535005C (en) | 2007-02-06 | 2007-02-06 | Long chain human insulin-like growth factor(LR3IGF-1) and its preparing process and application method |
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| CNB2007100034188A CN100535005C (en) | 2007-02-06 | 2007-02-06 | Long chain human insulin-like growth factor(LR3IGF-1) and its preparing process and application method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101172091B (en) * | 2007-09-25 | 2011-04-27 | 北京美福源生物医药科技有限公司 | Preparation process and application of fusion protein skin care product containing human serum albumin and skin cell growth factor |
| CN102776259A (en) * | 2011-05-13 | 2012-11-14 | 吴江近岸蛋白质科技有限公司 | Preparation method of long insulin-like growth factor 1 mutant |
-
2007
- 2007-02-06 CN CNB2007100034188A patent/CN100535005C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101172091B (en) * | 2007-09-25 | 2011-04-27 | 北京美福源生物医药科技有限公司 | Preparation process and application of fusion protein skin care product containing human serum albumin and skin cell growth factor |
| CN102776259A (en) * | 2011-05-13 | 2012-11-14 | 吴江近岸蛋白质科技有限公司 | Preparation method of long insulin-like growth factor 1 mutant |
| CN102776259B (en) * | 2011-05-13 | 2016-04-20 | 吴江近岸蛋白质科技有限公司 | The preparation method of long type-1 insulin like growth factor mutant |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100535005C (en) | 2009-09-02 |
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